The long term goals of my research are to study the basic mechanisms of formation, elimination and plasticity of synapses. One of the most important structures at the synapses is the site of transmitter release: the active zone. Under this proposal frog neuromuscular junctions will be studied in various situations using intracellular recording and freeze-fracture electron microscopy. The major objectives are to study: (I) Cytochemistry and differentiation of the active zone membranes. Filipin has been used as a cytochemical probe for membrane cholesterol. Distinctive filipin-sterol complexes are seen in most areas of freeze-fractured presynaptic membranes but are absent from active zones. Differentiation of this membrane heterogeneity, especially in relation to active zone particles and junctional folds, will be studied by treating neuroomuscular junctions with filipin during degeneration, regeneration and development. Changes in membrane heterogeneity at multiply-innervated endplates will also be examined, particularly during elimination of synapses in young frogs. Effects of cholesterol on transmitter release and active zone cytochemistry will be studied by applying cholesterol-rich liposomes to normal junctions. (II) Influence of postjunctional folds on the formation of active zones (1) Ectopic junctions will be induced de novo by removing the original endplate zone and implanting original nerves to enplate-free areas. Formation of active zones at ectopic sites with no previous junctional folds will be examined and compared with regeneration of active zones at original endplates with persisting junctional folds. (2) Vagus-Muscle junctions will be formed by implanting vagus nerves to denervated skeletal muscles. At these junctions it will be examined whether active zones regenerate to their original structures found at normal vagal terminals or differentiate into two double rows of particles as seen in motor nerve terminals. The proposed research will elucidate how the unique organization and cytochemistry of active zones is induced, differentiated and maintained. It will also provide a better understanding of the fundamental process of neurotransmitter release. This basic knowledge will have a strong impact on understanding the mechanisms of learning, intelligence development, and certain developmental neurological disorders.